Determination of toxicity

ABSTRACT

There is disclosed a method of determining the toxicity of a fluid sample comprising the steps of: (i) mixing the sample with a suspension of light emitting organisms; (ii) monitoring the light output (E) of the mixture continually over a period of time (t o  -t n ) using a photodetector device sensitive to the wave length of the emitted light; and (iii) determining the differential d(log E)/dt to give a measure of toxicity concentration.

This invention relates to a method of monitoring the toxicity of a fluidsample by means of measuring the light output of light emittingmicroorganisms which have been exposed to the sample. The metering andmixing of the sample medium with a fluidic suspension of the saidmicroorganisms is controlled automatically and the resulting lightoutput interpreted using kinetic theory to relate these data toparameters of toxicolo- gical significance, such as the EC₅₀ value, andthereby provide a quantitive estimate of the sample toxicity.

A number of procedures for the determination of toxicity usingluminescent bacteria are well known. For example, one such procedureinvolves the addition of a suspension of the bacterium of the genusPhotobacterium Phosphoreum to a number of serial dilutions of an aqueoussample. Each diluted aliquot is measured in sequence for the light fluxdue to P. Phosphoreum metabolism at two preset time intervals. A blank,containing no portion of the sample is included for comparison. Thelight emission, sample concentration, and time data are used to estimateparameters of toxicological significance, such as the well known EC₅₀,using a predetermined mathematical algorithm. It is well known that suchmeasurements must be carried out at constant temperature, such that themonitoring apparatus supporting the samples and their dilutions isthermostatically controlled. The bacterium being a naturally occurringspecies in sea water, requires careful adjustment of sample salinity tomaintain constant ionic strength, and thereby constant osmoticconditions, throughout the sample dilution aliquots.

Such toxicity testing is usually applied to aqueous samples, where only0.5 cm³ of sample is required for a typical test. The bacterium is usedas a liquid suspension reagent that is generated daily by reconstitutionfrom a freeze-dried standard product. The popular embodiment of such ameasurement device relies upon intensive manual manipulation of sample,osmotic adjuster and bacterium suspension aliquots. It is typical forthe sample cuvettes to be loaded manually into a detection devicesensitive to the wavelength of light emitted from the bacteria in eachtest suspension. A common feature of these procedures is the problemassociated with other properties of the sample solution which interferewith the detection of the emitted light. Examples of such propertiesinclude samples which are coloured or turbid. In this way the lightemission from the admixture between the test sample and the bacteriumsuspension reagent may be attenuated and lead to false estimates ofsample toxicity. Techniques to overcome the effects of the abovementioned attenuation usually involve additional equipment, such asmultichambered measurement cuvette, and additional complexity in respectof the optical measurement apparatus and data interpretation procedures.

The present invention is directed towards toxicity testing, employingthe well known principle of utilising light emitting organisms inconjunction with kinetic rate theory to provide a measurement approachthat reduces the complexity of the toxicity assay and at the same timeenhances measurement precision. A further feature is that the kineticrate approach affords a superior means of fully automating toxicitytesting, based upon light emitting organisms.

According to the present invention there is provided a method ofdetermining the toxicity of a fluid sample comprising the steps of:

(i) mixing the sample with a suspension of light emitting organisms;

(ii) monitoring the light output (E) of the mixture continually over aperiod of time (t_(o) -t_(n)) using a photodetector device sensitive tothe wavelength of the emitted light; and

(iii) determining the differential ##EQU1## to give a measure oftoxicity concentration.

The method of determining the toxicity of a fluid sample may comprisethe further steps of:

(iv) mixing a fluid sample free from toxicity with the solutioncontaining a suspension of light emitting organisms;

(v) monitoring the light output (B) of the mixture continually over aperiod of time of equal duration to the period (t_(o) -t_(n)) todetermine the decay in light output due to natural and environmentalfactors, and

(vi) using the values of light output (B) to obtain a corrected lightoutput value (E') for use in step (iii).

The steps (iv) and (v) may be carried out simultaneously with steps (i)and (ii) and corrected values of light output (E') may be obtained byusing the equation:

    E'.sub.t =E.sub.t /B.sub.t normalized

Alternatively the steps (iv) and (v) may be carried out to give lightoutput values (B1) before steps (i) and (ii) and again after steps (i)and (ii) to give light output values (B2) and wherein corrected valuesof light output E' are obtained using the equation: ##EQU2##

The light emitting organism may be the bacterium PhotobacteriumPhosphoreum.

The fluid sample may comprise municipal or industrial effluent. Anegative value for the differential d(logE')/dt indicates aconcentration of substance toxic to the organism and provides anindicator to possibly unacceptable levels of cocentration.

The invention will be further apparent from the following descriptionwith reference to the several figures of the accompanying drawings,which show, by way of example only, one form of apparatus for performingthe method thereof.

OF THE DRAWINGS:

FIG. 1 shows a graph of light output against time illustrating thetypical behaviour of light emitting bacteria under toxic load (a) andunder blank sample conditions (b);

FIG. 2 shows a graph of the logarithm of light output against time againunder toxic load (a) and under blank sample conditions (b);

FIG. 3 shows a graph of the slope of plots of the logarithm of correctedlight output against time against toxic concentration;

FIG. 4 shows a schematic diagram of the apparatus; and

FIG. 5 shows a number of possible flow cell designs for use in theapparatus of FIG. 4.

The method is applied to the toxicity testing of a fluid sample, throughadmixture with a suspension of light emitting organisms. The emittedlight is monitored with respect to time (see FIG. 1) in order to deducean algorithmic relationship between the decay in light output as afunction of both time and sample toxicity, such that sample toxicity maybe estimated. The method is based upon the fundamental assumption thatduring the monitoring period, the concentration, C_(T), of the toxicagent in any sample remains constant. It is also assumed that thesubsequent behaviour of the light emitting organisms may be described bythe process: ##EQU3##

It is also assumed, on the timescale of the typical measurementprocedure and over toxic concentration ranges that are not so great asto destroy the organism instantly with respect to that timescale, thatthe reaction follows pseudo first-order kinetic behaviour, with a rateconstant K_(r), which is independent of the concentration of theorganisms in the sample, suspended reagent admixture. The rate equationis well known: ##EQU4## where t is time, a is the initial light output,and x the decrease in light output. A plot of log (a-x) or (E) against twill yield a straight line of slope K_(r) (FIG. 2). K_(r) is related tothe reaction half-life, t₀.5, by ##EQU5##

The reaction half-life corresponds with the EC₅₀ condition, and maythereby be deduced through a priori calibration of the measurement witha standardized toxic agent (e.g. 3,5-dichlorophenol). A further featureof the behaviour of light emitting organisms, is that they will exhibita natural decay in light output even in the absence of a toxic agent. Toa first approximation this may be represented by a small negative slope,under the same measurement algorithm as used for toxicity testing, asshown in FIGS. 1b and 2b. This background, or blank decay, may thus becompensated for, to give corrected light emmission values (E') through aprocess of separate measurement of a standard blank in conjunction withmeasurement of a sample undergoing toxicity testing.

FIG. 3 shows the relationship between the differential ##EQU6## andtoxicity concentration. A negative value for the differential indicatesa concentration toxic to the organism and provides an indicator topossibly unacceptable concentrations of toxicity for many requirements.

Referring now to FIG. 4, it will be seen that the apparatus essentiallycomprises a dual channel fluid flow analysis manifold which can receivesample to be tested, a diluent, the bacterium suspension and a calibrantthrough a series of valves V in required proportions to each of firstand second flow cells F1 and F2 monitored by photodetectors P1 and P2reporting to a microprocessor C.

The sample, either as a continuous stream, as might be obtained from asuitable sampling device, or as a batch sample, which may be deliveredmanually or as a series of samples by use of an autosampler device forexample, is introduced into the flow manifold. The manifold may beconstructed from tubes or conduits of a variety of materials with arange of dimensions to control the flow and transport characteristics ofthe sample introduction. Similarly, the diluent, such as a salinesolution, may be added to the sample in a precise way. The sample streammay therefore be transported without modification or with apredetermined dilution factor. The diluent also serves as an inertmedium to sequentially carry discrete sample volumes through themanifold. A further feature of the diluent is its application as a blanksample for calibration purposes. Furthermore, excess diluent may be usedto flush and thereby wash the manifold between assays and as required.It is evident that although in this example the diluent ismultifunctional, it would be possible to introduce further reservoirs,linked with the appropriate valves or diverters, for the operations ofblank calibration and washing.

The flow cells should be constructed of material which is transparent tothe wavelength of the light emission of the particular microorganismsemployed. It is obvious that the flow cell may be of such a volume as tocontain the entire admixture or only a portion of the light emission. Itis preferred that the admixture is quiescent within the flow cell duringthe kinetic monitoring period, to maintain best optical precision of thelight emission measurement.

The entire manifold, including reagent and sample reservoirs, should bemaintained at constant temperature. Any temperature may be utilized,providing that it remains within the operating range of themicroorganisms and does not adversely affect liquid viscosity within themanifold. Temperature control may be achieved through any knownrecognized means.

Although this preferred embodiment utilises liquid transportationthrough the drawing of liquids through a manifold by means of anejector, it is clear that many alternatives may be invoked to achievethe said aim of liquid transportation. For example, the liquidreservoirs may be pressurised to force the liquid through the manifold.Alternatively pumping devices may be introduced into some or all of themanifold branches. Furthermore, in miniature form, electroosmotictransport could be utilized. Other embodiments could utilise flow bygravity, capillary action or centrifugal motion.

The outputs from the optical detectors are electronically modified bysignal processors to generate a voltage or current suitable fordigitisation into the microprocessor. Alternatively analogue computationmay be employed, but a digital means is preferred. The entiremeasurement operation, including selection of manifold configurationwithin a timed sequence, and monitoring of the function of lightemission with time, may all be coordinated using the microprocessor C.The principle of biokinetic monitoring described above, may beimplemented through a suitable algorithm introduced into themicroprocessor C. In this way a read-out is produced to convey thedesired information and measured parameters obtained from the toxicassay operation. Further status information may be advantageouslygenerated for the information of the operator.

Referring to FIG. 5, four possible designs of flow cell are shown. Eachflow cell has a liquid input and output, combined with means forinterfacing the light emission from the admixture in the cell volumewith the optical detectors. The cell shown in FIG. 5(a) comprises asingle tube constructed from optically transparent material. Thedimensions of length and internal cross-section may be selected togenerate a cell volume that is compatible with the admixture volumeunder study. The cross-section may be of any geometrical design, butcircular or rectangular cross-section is preferred.

FIG. 5(b) depicts a flow cell, similar to that of FIG. 5(a), but withthe addition of a thin layer portion, designed to increase the emissionarea of the admixture. This design would be particularly appropriate forhighly coloured samples, whereby the absorbance path is minimized.

FIG. 5(c) depicts a spiral flow cell, which is based upon the propertiesof flow cell 5(a). The spiral design enables a greater admixture volumeto be maintained within the active area boundary of the opticaldetection device, whilst maintaining flow in narrow bore tubes. Narrowbore tubes generally maintain the integrity of the admixture plugthrough reduced dispersion.

FIG. 5(d) depicts a thin layer wall-jet flow cell, comprising a flat,circular optical window, W1, mounted within a cell body with the meansof a narrow bore jet inlet and a low flow restriction output.

Valve V1 selects either pure sample, pure diluent or mixes a controlledportion of sample to form a diluted admixture. Valve V2 diverts diluentto either branch of the dual channel instrument. Valve V3 enables abackground admixture, between the bacterium suspension and diluent to beformed. Valve V4 diverts the bacterium suspension to either branch ofthe dual channel instrument, thus providing luminescent reagent for bothtoxic and background decay curve monitoring. Valve V5 generates anadmixture between the bacterium suspension and either diluent or sampleor a sample/diluent admixture. Alternatively, valves V5 and V1 may bedeactivated to provide a constant flow between the diluent reservoir andflow cell for the purpose of washing. In a similar fashion, with valveV3 deactivated and valve V2 activated, flow cell 2 may also be washed.Valve V6 selects the outflow from either flow cell 1 or flow cell 2.Valve V7 enables the selection of either the flow cell effluent, or aninput of ambient air. This enables the manifold flow to be halted forextended periods, as required. An ejector, powered either from a wateror air source, provides the preferred means of drawing liquids throughthe manifold, and in conjunction with any flow pattern that is set upthrough the control of the diverter valves. Valve V8 enablesintroduction of a calibrant.

Detectors P1 and P2 monitor the light emission (E) from the sample undertest and that (B) from an admixture of diluent and bacterium suspensionalone to determine the background decay of light emission.

The microprocessor C computes corrected values E' using the formula:##EQU7##

The values B are normalised by dividing all data points by the firstdata point in the sequence.

The differential ##EQU8## is then calculated to give a measure oftoxicity.

In many applications if the value of the differential is negative thatis indicative of an unacceptable level of toxicity and an alarm signalmay be generated.

It will be appreciated that it is not intended to limit the invention tothe above example only, many variations, such as might readily occur toone skilled in the art, being possible, without departing from the scopethereof as defined by the appended claims.

We claim:
 1. A method of determining the toxicity of a fluid samplecomprising the steps of:(i) mixing the sample with a suspension of lightemitting microorganisms; (ii) monitoring the light output (E) of themixture continually over a period of time (t_(o) -t_(n)) using aphotodetector device sensitive to the wavelength of the emitted light;and (iii) determining the differential d(log E)/dt to give a measure oftoxicity concentration.
 2. The method of determining the toxicity of afluid sample according to claim 1 further comprising the steps of:(iv)mixing a fluid sample free from toxicity with the solution containing asuspension of the light emitting microorganisms; (v) monitoring thelight output (B) of the mixture continually over a period of time ofequal duration to the period (t_(o) -t_(n)) to determine the decay inlight output due to natural and environmental factors, and (vi) usingthe values of light output (B) to obtain a corrected light output value(E') for use in step (iii).
 3. The method according to claim 2 whereinsteps (iv) and (v) are carried out simultaneously with steps (i) and(ii) and wherein corrected values of light output (E') are obtained byusing the equation:

    E'.sub.t =E.sub.t /B.sub.t normalized.


4. The method according to claim 2 wherein steps (iv) and (v) arecarried out to give light output values (B1) before steps (i) and (ii)and again after steps (i) and (ii) to give light output values (B2) andwherein corrected values of light output E' are obtained using theequation: ##EQU9##
 5. The method according to claim 1 wherein the lightemitting microorganism is the bacterium Photobacterium phosphoreum. 6.The method according to claim 5 wherein the fluid sample comprisesmunicipal or industrial effluent and wherein an unacceptable level oftoxicity is recognized by a negative value for the differential##EQU10##
 7. The method according to claim 2 wherein the light emittingorganism is the bacterium Photobacterium phosphoreum.